Effects of Different Tissue Factor and Phospholipid Concentrations on Thrombin Generation Assay Parameters in Normal...

Thrombin generation assay (TGA) is an assay that activates the coagulation cascade using a trigger reagent containing tissue factor (TF) and phospholipids (PL), and the generated thrombin is detected using a fluorogenic substrate. The standardisation of assay conditions and validation of in-house methods are recommended for research investigations. This study aimed to investigate the influence of TF and PL concentrations on TGA parameters in normal and abnormal plasma samples and the usefulness of commercial quality control materials for standardisation.

Five samples were employed for the TGA: Pooled Normal Plasma and Ci-Trol 1 as normal samples as well as Ci-Trol 2, Ci-Trol 3 and 2.5-fold diluted Pooled Normal Plasma. Trigger reagents were prepared with various TF and PL concentrations. The final TF concentrations were 0-50 pM with 4 μM PL, while the final PL concentrations were 0-40 μM with 5 pM TF. The lag time, peak height, time-to-peak and endogenous thrombin potential were calculated.

The lag time and time-to-peak prolonged, and the peak height decreased as TF concentration decreased. In contrast, PL concentration primarily affected peak height. Clear thrombin peaks were confirmed in Ci-Trol 2 and Ci-Trol 3.

Changes in TF concentration affected the lag time time-to-peak, and peak height, while alterations in PL concentration primarily affected the peak height in both normal and abnormal plasma samples. These findings are crucial for assay standardisation; Ci-Trol 2 and Ci-Trol 3 may serve as quality control materials.